Bull Vet Inst Pulawy 49, 455-464, 2005
EFFECT OF AGED GARLIC EXTRACT AND ALLICIN ADMINISTRATION TO SOWS DURING PREGNANCY AND LACTATION ON BODY WEIGHT GAIN AND GASTROINTESTINAL TRACT DEVELOPMENT OF PIGLETS: MORPHOLOGICAL PROPERTIES OF THE SMALL INTESTINE. PART II. MARCIN R. TATARA, EWA ŚLIWA, KATARZYNA DUDEK, SYLWESTER KOWALIK, ANTONI GAWRON1, TOMASZ PIERSIAK1, PIOTR DOBROWOLSKI,1 AND TADEUSZ STUDZIŃSKI Department of Animal Physiology, Faculty of Veterinary Medicine, Agricultural University of Lublin, 20-950 Lublin, Poland 1 Department of Comparative Anatomy and Anthropology, Maria Curie-Skłodowska University, 20-950 Lublin, Poland e-mail:
[email protected] Received for publication August 02, 2005.
Abstract This investigation was undertaken to continue our earlier studies performed on piglets born by sows that were treated with aged garlic extract (AGE) and allicin during the last 24 d of pregnancy and during 28 d of lactation. To explain whether improved systemic development of piglets from sows treated with AGE or allicin observed previously is connected with changes of the gastrointestinal tract on microstructural level, the morphological properties of the small intestine were determined. Piglets were obtained from 18 sows and divided into 3 equal experimental groups. The experimental piglets were divided additionally into 8 age-differentiated subgroups, namely non-suckling newborns and 1, 3, 7, 14, 28, 35, and 56 d old piglets. Starting from the 91st d of pregnancy up to the piglet weaning on the 28th d of their life, the sows were daily given per os AGE or allicin, whereas the control group received the vehiculum. Morphometric measurements of villus height, villus width at villus base, villus section area, crypt (gland) depth and mucosa thickness were performed automatically under confocal microscope. Moreover, villus number per cm of cross section of each investigated intestine sample was estimated. This study revealed that administration of allicin and AGE to pregnant and lactating sows induced beneficial effects on morphological parameters of villi investigated in different parts of the small intestine and during various developmental stages of their offspring. These effects seem to be dependent on the age of the animals and the examined part of the small intestine. The most readable changes in villus morphology were induced by AGE and allicin in piglets between 7 and 35 d of life. Moreover, the most significant response of the intestine to experimental factors was observed in 50% and 75% of jejunum length as well as in the ileum. In conclusion, increased surface of the small intestine, as a positive consequence of allicin and AGE administration may be postulated as factors responsible for their improved systemic development. Moreover, the results obtained confirmed that improved nutrition at early stages of
postnatal development, and as the major intrauterine environmental factor, may be beneficial not only after the birth but may have lifelong consequences, leading to the permanent changes of the structure, physiology, and metabolism of offspring.
Key words: sows, piglets, aged garlic extract, allicin, small intestine. Beneficial effects of garlic (Allium sativum) on an organism arise from a wide variety of components that may act synergistically. The primary sulphurcontaining constituents in intact garlic cloves are the γglutamyl-S-alk(en)yl-L-cysteines and S-alk(en)yl-Lcysteine sulphoxides, including alliin. Additional constituents of intact garlic cloves include steroidal glycosides, lectins, prostaglandins, fructan, pectin, essential oil, adenosine, vitamins B1, B2, B6, C, E, biotin, nicotinic acid, fatty acids, glycolipids, phospholipids, anthocyanins, flavonoids, phenolics and essential amino acids (1, 5, 8, 9). Positive effects of garlic compounds include reduction of risk factors for cardiovascular diseases and cancer, stimulation of immune functions, detoxification, radioprotection, restoration of physical strength, resistance to various kinds of stress and potential antiaging effects (1, 3, 13). Available garlic products can be classified into 4 groups, i.e. essential oil, garlic oil macerate, garlic powder and garlic extract. The extract, especially aged garlic extract (AGE), contains mainly water-soluble constituents from garlic and a small amount of oil-soluble compounds (14). Hoshino et al. (7) reported that in contrast to dehydrated raw garlic powder and dehydrated boiled garlic powder, AGE did not cause any undesirable effects on the gastrointestinal
456 mucosa. Furthermore, aging was postulated as the most effective methods for the elimination of toxic compounds of raw garlic that are responsible for its negative effects observed in the gastrointestinal tract of dogs (7). The most important sulfur compounds of AGE are S-allyl cysteine (SAC) and S-allyl mercaptocysteine (10). The bioavailability of SAC is about 100% in mice, 98.2% in rats and 87.2% in dogs. Allicin, as an oil soluble compound of garlic is present in oil macerate. Similarly to other oil-soluble compounds like sulphides, ajoene, and vinyldithiins, allicin was not found in blood or urine 1 h after garlic consumptions which indicates its rapid conversion to diallyl disulphide (1). Next to the systemic effects of garlic compounds, antibacterial and antifungal effects of allicin and AGE were reported (1, 2, 11). According to our earlier studies that showed positive influence of the aged garlic extract and allicin on the immune system of piglets and sows as well as on gastrointestinal tract development in piglets (3, 12, 13), we aimed to investigate the effect of allicin and AGE administration to pregnant and lactating sows on villus morphology in several small intestine segments of their piglets.
Material and Methods Experimental design and sampling procedure. The experimental procedures used throughout this study were approved by the Local Ethics Committee on Animal Experimentation of the Agricultural University of Lublin, Poland. The experiment was performed on piglets born by sows of the Large Polish White breed. The sows, starting from day 91 of pregnancy up to piglet weaning on day 28 of their life, were daily given per os AGE (n=6) at the dosage of 10 ml/100 kg b.w. or allicin (Alliomax, Herbapol Lublin) (n = 6) at the dosage of 1.6 mg/100 kg b.w. Control sows (n = 6) received 10 ml/100 kg b.w. of vehiculum (soybean oil). The animals were kept under standard rearing conditions with free access to fresh water and fed a well balanced diet as described previously (12). The piglets were divided into 3 equal experimental groups. The piglets from AGE- or allicintreated sows were assigned to AGE and ALL groups, respectively, while the piglets born by sows that received vehiculum belonged to the control group (Con). The experimental animals were divided additionally into 8 age-differentiated subgroups (each subgroup comprised 6 animals), namely non-suckling newborns, and 1, 3, 7, 14, 28, 35, and 56 d old piglets. After the piglets were sacrificed at the mentioned above age, the small intestine was isolated, the mesentery was removed gently from the intestine and 1 cm in length transverse sections of the duodenum, jejunum (25%, 50%, and 75% of total small intestine length) and ileum were collected similarly for all samples. To avoid sex-conditioned differences of the small intestine morphology, the sacrificed piglets within age-differentiated subgroups were sex-matched. Morphological examination. The intestine samples were immediately fixed in fresh 4% buffered
formaldehyde, dehydrated in growing concentration of ethyl alcohol, cleared in xylene, and embedded in paraplast (Sigma-Aldrich). Serial sections were cut at 4.5 µm, stained with haematoxylin and eosin and closed with the use of DPX (Sigma-Aldrich). The samples were examined using light and confocal (AXIOVERT 200 M equipped with an LSM 5 Pascal laser scanning head, Zeiss) microscopes. Simultaneous collection of fluorescent and Nomarsky contrast images was performed using argon laser, with length wave of 514 nm. Morphometric measurements of villus height (VH), villus width at villus base (VW), villus section area (VSA), crypt (gland) depth (CD), and mucosa thickness (MT) were performed automatically under confocal microscope using LSM 5 Image Examiner software. Moreover, villus number per cm (VN) of cross section of each investigated intestine sample was estimated. Statistical analysis. Statistical analysis was performed using Statistica software (version 6.0). All the data were presented as means ± SEM. All the examined parameters were found to be normally distributed in accordance with Kolomogorov-Smirnov test. Differences between AGE or ALL group versus control group were tested for statistical significance with the use of Student’s ttest. Differences showing P ≤ 0.05 were considered significant.
Results Results of morphological examinations in several segments of small intestine of piglets are shown in Tables 1 – 5. Newborn piglets from ALL group reached higher value of VN in the duodenum, jejunum (25% and 50% of its length), and ileum, when compared to the control group. The piglets had increased MT in the jejunum (50% and 75% of its length) and ileum and increased VH and CD in 75% of jejunum length. Moreover, VSA was higher in the ileum. AGE administration caused significantly higher values of VH, VSA, and MT in 75% of jejunum length and in the ileum, as well as CD value in 75% of jejunum length in newborn piglets. The results of histological examination of the intestine samples from the duodenum, 50% of jejunum length and ileum of newborn piglets from control sows and from those treated with AGE and allicin are presented on Fig. 1. One day old piglets from AGE group reached higher value of VW and CD in 25% and 50% of jejunum length. Except for VH, all other investigated parameters in 75% of jejunum length were increased in this group. Similar changes were observed in these animals in the ileum, when VH, VSA and MT were analysed. Analysis of intestine structure in 1 d old piglets from ALL group revealed increased VN in the jejunum (50% and 75% of its length) and ileum. Moreover, these piglets showed higher values of VH and VSA in 75% of jejunum length, when compared to the controls.
457 Con
AGE
ALL
A)
B)
C)
Fig. 1. Confocal microscopy of intestine samples from the duodenum (A), 50% of jejunum length (B) and ileum (C) of newborn piglets from control (Con) sows and from those treated with aged garlic extract (AGE) and allicin (ALL)(100x).
458 Three day old piglets from mothers treated with AGE reached higher values of VH, VSA, MT and VN in the duodenum, when compared to the control group. In 50% of jejunum length, piglets from the AGE group reached significantly higher values of VSA. Analysis of 75% of jejunum length showed that AGE administration to pregnant and lactating sows significantly increased VH, VW, VSA, CD, and MT in 3 d old piglets. Allicin administration caused higher values of VH and MT in the duodenum of 3 d old piglets in comparison to controls. Analysis of VH, VW, VSA, CD, and MT in 50% of jejunum length showed higher values in piglets from ALL group. Furthermore, higher value of VSA in 75% of jejunum length was observed in these piglets, when compared to the control group. AGE administration increased VH, VSA, MT, and VN of the duodenum and CD, MT, and VN in 25% of jejunum length in 7 d old piglets. Except for CD, all other investigated parameters in 50% of jejunum length were significantly higher, when compared to the controls. Moreover, the piglets had higher values of VH and MT in 75% of jejunum length and MT in the ileum. At the age of 7 d, piglets from the ALL group reached higher values of VH, VW, CD, and VN in 25% of jejunum length, when compared to the control group. Similar changes were observed in 50% of jejunum, length when analysing VH, MT and VN. VN in 75% of jejunum length and MT in the ileum were also significantly increased in piglets from the ALL group. Examination of 14 d old piglets showed that both AGE and allicin administration increased VH and VN in the duodenum, when compared to the control group. Similar effect of these substances was observed in all parts of the jejunum, when analysis of VH and MT was performed. Moreover, CD, VSA, and VH values increased in 50% and 75% of jejunum length and in the ileum after AGE and allicin treatment. Fourteen day old piglets from AGE group had significantly higher values of VW and VSA in 50% of jejunum length and VSA in the ileum, whereas allicin administration increased VN in the jejunum (50% and 75%) and ileum. Twenty eight day old piglets from AGE group reached higher values of CD and MT in the duodenum, whereas allicin administration increased significantly CD and VN, when compared to the controls. Piglets from AGE and ALL groups at the weaning time reached higher values of CD and MT (25% and 75% of jejunum length), VW (50% of jejunum length), and VH, VW, VSA, CD, and MT in the ileum. Furthermore, AGE treatment increased VSA in all parts of the jejunum as well as VH and VW in 75% of its length. The analysis of morphological properties of the small intestine in 35 d old piglets revealed higher values of all investigated parameters in the duodenum after AGE treatment, while allicin increased only VW and MT in this part of the intestine. Piglets from the AGE group reached higher values of VH, VW, CD, and VN in 25% of jejunum length. On the other hand, values of VH, VW, VSA, and MT increased in this part of the intestine in piglets from the ALL group. AGE and allicin administration increased VH, VSA, and MT in 50% of jejunum length and ileum as well as VH in 75% of jejunum length and CD in the ileum.
Piglets from AGE group reached higher values of CD in 50% of jejunum length, VW and VSA in 75% of its length and VN in the ileum. Moreover, allicin treatment enhanced VW value in 50% of jejunum length in piglets 1 week after weaning. Fifty six day old piglets from both AGE and ALL groups reached significantly higher values of VSA and MT in the duodenum. AGE administration increased VN in 25% of jejunum length and CD in 50% of its length; however, piglets from ALL group had higher values of VW in 25% of jejunum length and VN in 50% of its length. The analysis of the ileum showed that VN and MT increased in piglets from AGE and ALL groups. However, significantly higher values of VH were obtained in both experimental groups, when compared to the controls.
Discussion Our earlier studies performed on pigs showed that AGE and allicin administration to pregnant and lactating sows induces positive effects on body weight determined at birth and during 56 d of postnatal development of piglets. Next to effects on body weight gain induced by AGE and allicin, stimulation of systemic development of piglets by these substances was proven by higher values of internal organs like the liver, pancreas, stomach, as well as increased small intestine length (12). This investigation was undertaken to continue our earlier studies, and explain whether results obtained previously are connected with microstructural properties of the small intestine. Results obtained in this study showed different morphology of the small intestine at microstructural level in age-differentiated piglets from all groups of the experiment. Thus, to facilitate the analysis of effects induced by the administration of AGE and allicin to pregnant and lactating sows on microstructural properties of the small intestine in their piglets, all the investigated parameters of villus morphology should be considered. This study revealed that administration of allicin and AGE to pregnant and lactating sows induced beneficial effects on morphological parameters of villi investigated in different parts of the small intestine and during various developmental stages of their offspring. These effects seem to be dependent on the age of animals and the examined part of the small intestine. It is noteworthy that the most readable changes in villus morphology were induced by AGE and allicin in piglets between 7 and 35 d of life. Moreover, the most significant response of the intestine to experimental factors was observed in 50% and 75% of jejunum length as well as in the ileum. These data suggest that enhanced systemic development of piglets reported previously may be achieved by increased intestine development observed at macro- and microstructural level. These results are generally in accordance with investigation performed by Hoshino et al. (7), where AGE was postulated as the most suitable form of garlic preparation for long-term oral administration.
459
Table 1 Morphometric measurements of villus height (VH), villus width (VW), villus section area (VSA), crypt depth (CD), mucosa thickness (MT) and villus number per cm (VN) of cross section of the duodenum in the newborn piglets and at the age of 1, 3, 7, 14, 28, 35, and 56 d from control (Con) sows and from those treated with aged garlic extract (AGE) and allicin (ALL) during pregnancy and lactation VH (µm) Age (d) Newborns 1 3 7 14 28 35 56
VSA (µm2)
VW (µm)
Con
AGE
ALL
Con AGE ALL
Con
CD (µm)
AGE
ALL
MT (µm)
Con AGE ALL
VN
Con
AGE
ALL
Con AGE
ALL
557
401*
243*
83
70*
60*
32052
25688
16023*
107
110
118
693
527*
418*
100
86
133*
± 35
± 28
± 25
±1
±4
±1
± 2549
± 2712
± 1260
±7
±6
±3
± 37
± 36
± 26
±6
±1
±4
578
437*
520
79
88
67
51090
30965*
44925
171
165
147*
884
635*
772*
95
91
98
± 42
± 48
± 30
±6
±4
±4
± 5302
± 3700
± 3027
±7
±4
±2
± 28
± 31
± 15
±4
±2
±4
513
892*
653*
130
95*
117
47884
92381*
55258
297
129*
242
695
91
101*
102
± 19
± 52
± 30
±5
±3
±7
± 3891
± 7067
± 1344
± 46
± 66
± 14
± 46
± 23
± 33
±3
±2
±6
397
1176*
479
114
99
118
38259 112545*
33981
305
203* 226*
772
1543*
644
70
99*
79
± 47
± 47
± 38
±7
±3
±2
± 3990
± 3138
± 11
±9
±9
± 64
± 107
± 47
±2
±6
±5
79*
± 3960
1085* 889*
420
620*
584*
184
110
118
58511
58418
56567
310
203*
276
812
852
784
62
98*
± 30
± 40
± 18
± 37
±6
±4
± 6714
± 4492
± 2362
± 15
±5
±7
± 30
± 27
± 19
±2
±2
±1
353
328
322
121
124
100
46542
44773
39683
229
382* 270*
638
784*
671
60
57
76*
± 31
± 24
± 15
±8
±3
±6
± 4411
± 2916
± 1227
± 11
± 29
±8
± 44
± 27
± 15
±4
±2
±2
294
735*
323
100
33465
78669*
29798
248
389*
270
530
1152* 582*
70
78*
68
± 53
± 40
± 36
±3
± 4949
± 3669
± 5998
± 36
± 41
± 17
± 16
±2
±3
±1
39916
41153
56498*
305
192*
269
± 4572
± 5771
± 5298
± 11
±4
± 18
283
327
342
160
± 24
± 49
± 14
±6
124* 135* ±8
±7
104* 120* ±4
±4
± 45
± 15
616
615
743*
57
61
57
± 26
± 25
± 32
±3
±5
±1
* P ≤ 0.05 versus control group.
459
460
460
Table 2 Morphometric measurements of villus height (VH), villus width (VW), villus section area (VSA), crypt depth (CD), mucosa thickness (MT) and villus number per cm (VN) of cross section of 25% of jejunum length in the newborn piglets and at the age of 1, 3, 7, 14, 28, 35, and 56 d from control (Con) sows and from those treated with aged garlic extract (AGE) and allicin (ALL) during pregnancy and lactation
Age (d) Newborns 1 3 7 14 28 35 56
VSA (µm2)
VH (µm)
VW (µm)
Con AGE ALL
Con AGE ALL
Con
AGE
ALL
CD (µm)
MT (µm)
VN
Con AGE ALL
Con AGE ALL
Con AGE
ALL
765
366*
733
95
57*
68*
56909
18322*
48100
99
110
92*
863
525*
877
99
98
± 26
± 23
± 14
±2
±2
±3
± 2566
± 1978
± 3638
±2
±9
±3
± 34
± 20
± 27
±3
±3
±5
895
897
913
68
90*
67
66545
75506
63957
139
124*
98*
884
635* 772*
99
89*
106
± 40
± 60
± 34
±5
±3
±2
± 4002
± 7808
± 4791
±4
±6
±4
± 28
± 31
± 15
±5
±2
±3
921
946
482*
107
97
85*
80907
89157
25967*
126
126
137
975
1096 672*
105
103
90
± 35
± 56
± 43
±5
±3
±4
± 6751
± 6401
± 1435
±4
±6
±6
± 53
± 30
±4
±3
±9
858
914*
957
89
125*
88
± 14
±9
± 46
±2
± 10
±4
± 26
122*
609
730
797*
84
89
120*
59490
65637
59332
126
± 64
± 27
± 28
±6
±3
±3
± 6275
± 2724
± 1547
±4
±6
±7
330
442* 577*
116
91*
88*
35097
32846
46480
167
160
135*
335
616* 761*
77
78
84
± 21
± 13
± 10
±4
±4
± 5483
± 2383
± 2191
±8
±6
±5
± 27
± 25
±6
±5
±3
± 18
159* 169*
± 16
343
383
280
95
101
92
27126
50997*
28427
167
292* 256*
522
679* 617*
91
66*
60*
± 30
± 34
± 12
± 11
±8
±3
± 2942
± 2557
± 2117
± 13
± 25
±8
± 10
± 43
± 17
±3
±4
±3
369
477* 531*
84
145* 114*
38826
40106
70164*
245
294*
245
677
634
818*
69
75*
71
± 25
± 10
±2
± 15
±3
± 3299
± 1295
± 3904
± 14
± 17
±9
± 37
± 25
± 23
±2
±2
±8
± 36
228
228
218
85
56*
131*
28294
28849
31478
209
207
218
540
531
557
70
79*
71
± 16
± 12
± 15
±6
±1
±6
± 1172
± 775
± 1387
± 11
± 10
± 15
± 23
± 30
± 43
±3
±3
±3
* P ≤ 0.05 versus control group.
461
Table 3 Morphometric measurements of villus height (VH), villus width (VW), villus section area (VSA), crypt depth (CD), mucosa thickness (MT) and villus number per cm (VN) of cross section of 50% of jejunum length in the newborn piglets and at the age of 1, 3, 7, 14, 28, 35, and 56 d from control (Con) sows and from those treated with aged garlic extract (AGE) and allicin (ALL) during pregnancy and lactation VH (µm) Age (d) Newborns 1 3 7 14 28 35 56
Con
AGE
VSA (µm2)
VW (µm) ALL
Con AGE ALL
Con
AGE
CD (µm) ALL
MT (µm)
Con AGE ALL
VN
Con AGE
ALL
Con AGE ALL
652
431*
659
73
56*
66
47110
24841*
50056
76
71
72
701
533*
833*
104
82*
± 12
± 34
± 24
±4
±4
±2
± 2872
± 1918
± 2840
±5
±2
±4
± 12
± 35
± 28
±3
±4
±3
640
609
716
73
80
47*
56587
52929
52567
102
116*
83*
816
772
880
93
91
125*
± 59
± 28
± 36
±4
±4
±2
± 4903
± 1760
± 3911
± 27
619
831
1246*
71
58*
120*
53719
78242* 100778*
± 56
± 117
± 58
±2
±3
±4
± 3749
± 8565
± 17831
242
783*
461*
83
100*
76
31028
51826*
28507
119*
±2
±5
±3
± 40
± 21
±2
±2
±5
109
103
170*
881
1025 1419*
102
99
100
±4
±4
± 11
± 64
± 115
± 50
±2
±2
±5
143
132
124
517
962*
614*
73
±3
105* 120*
± 66
± 38
± 44
±4
±3
±4
± 5100
± 1912
± 1097
±5
± 10
± 14
± 26
± 38
±2
±9
±5
375
479*
527*
90
117*
81
38515
55368*
46760
120
154* 139*
524
692*
733*
71
71
98*
± 16
± 32
± 21
±5
±4
±5
± 2255
± 4119
± 4455
±5
± 12
±6
± 28
± 22
± 25
±3
±5
±5
394
361
343
76
30804
51714*
28713
183
197
209
588
646
507
67
71
60
± 18
±8
± 34
±5
± 1488
± 5031
± 2298
± 10
± 16
± 10
± 42
± 23
± 25
±4
±3
±2
300* 191*
56*
117* 126* ±4
±5
265
340*
385*
101
123
120*
31941
43010*
38289*
257
560
686*
657*
67
72
± 14
± 30
± 22
±4
± 10
±7
± 1668
± 2804
± 2199
±8
±4
±8
± 25
± 22
± 33
±2
±1
±4
301
325
311
80
89
86
38438
43711
37809
195
227*
205
529
568
587
62
57
105*
± 14
± 34
±7
±4
±9
±3
± 1896
± 3874
± 3058
±8
± 11
±5
± 12
± 23
± 31
±1
±5
±6
* P ≤ 0.05 versus control group.
461
462 462
Table 4 Morphometric measurements of villus height (VH), villus width (VW), villus section area (VSA), crypt depth (CD), mucosa thickness (MT) and villus number per cm (VN) of cross section of 75% of jejunum length in the newborn piglets and at the age of 1, 3, 7, 14, 28, 35, and 56 d from control (Con) sows and from those treated with aged garlic extract (AGE) and allicin (ALL) during pregnancy and lactation VH (µm) Age (d) Newborns 1 3d 7 14 28 35 56
VSA (µm2)
VW (µm)
Con
AGE
ALL
Con AGE ALL
Con
AGE
CD (µm) ALL
MT (µm)
Con AGE ALL
VN
Con
AGE
ALL
Con AGE
ALL 107
462
800*
662*
59
63
66
33027
55265*
40935
62
80*
80*
567
908*
780*
107
94
± 44
± 19
± 32
±2
±2
±4
± 3733
± 2843
± 1662
±5
±4
±3
± 16
± 20
± 32
±6
±5
±2
668
750
831*
73
94*
71
51105
71706*
68613*
91
122*
91
830
980*
953
89
104*
94*
± 61
± 30
± 34
±2
±3
±3
± 3770
± 3239
± 5486
±5
±3
±4
± 57
± 32
± 57
±3
±4
±2
696
937*
768
73
98*
85
58487
98168*
76485*
105
132*
119
864
1092*
944
91
86
97
± 37
± 34
± 22
±2
±4
±5
± 2461
± 2704
± 1397
±2
±3
±8
± 42
± 40
±7
±3
±2
±4
105*
664
1353*
520
90
105
80
96799
112450
95304
126
128
143
401
1581*
622
84
89
± 82
± 52
± 46
±8
±3
±2
± 7357
± 3982
± 4934
±8
±6
±7
± 16
± 24
± 32
±3
±2
±3
181
661*
726*
112
110
113
19038
62675*
79867*
203
1076* 902*
71
75
100*
± 12
± 16
± 32
±7
±4
±7
± 2531
± 3377
± 8853
±7
± 10
± 15
± 85
± 39
±3
±2
±3
241
339*
251
81
102*
73
21619
30979*
24179
156
197* 208*
434
576*
497*
65
64
68
±5
± 17
±9
±6
±8
±5
± 1540
± 1408
± 1402
±4
± 10
± 12
±5
± 30
± 22
±3
±4
±2
210
282*
262*
99
118*
109
26916
31559*
23820
204
189
157*
458
475
469
61
64
72
± 17
±5
± 12
±5
±7
±6
± 1521
± 1355
± 2059
±5
±8
±8
±8
± 10
± 17
±4
±2
±4
178* 155* ±7
360
273
250
275
142
113
126
41127
35023
46041
232
206
269
556
500
624
54
63
55
±5
± 21
± 21
± 10
±7
±6
± 1154
± 3909
± 2187
± 14
± 20
± 18
± 25
± 10
± 21
±3
±3
±3
* P ≤ 0.05 versus control group.
463
Table 5 Morphometric measurements of villus height (VH), villus width (VW), villus section area (VSA), crypt depth (CD), mucosa thickness (MT) and villus number per cm (VN) of cross section of the ileum in the newborn piglets and at the age of 1, 3, 7, 14, 28, 35, and 56 d from control (Con) sows and from those treated with aged garlic extract (AGE) and allicin (ALL) during pregnancy and lactation
Age (d) Newborns 1 3 7 14 28 35 56
VSA (µm2)
VH (µm)
VW (µm)
Con AGE ALL
Con AGE ALL
CD (µm)
Con
AGE
ALL
Con AGE
MT (µm)
VN
ALL
Con AGE ALL
Con AGE ALL
404
789*
489
85
66*
65*
28907
60036*
36706*
129
93*
128
562
975* 694*
83
91
± 10
± 37
± 57
±4
±1
±2
± 1156
± 3320
± 3119
±2
±1
± 20
±3
± 38
± 53
±1
±3
±4
369
699*
407
103
85*
66*
37788
64559*
33399
172
131*
108*
602
911*
834
79
90
94*
± 62
± 118
± 80
±5
±6
±3
± 6204
± 9268
± 3184
±6
±3
±7
± 65
± 85
± 182
±4
±5
±5
390
306
457
104
87*
88*
26666
23922
29044
199
132*
115*
624
442*
610
81
88
92
± 12
± 40
± 47
±3
±5
±5
± 680
± 2261
± 3017
± 19
±5
±4
± 12
± 48
± 49
±6
±3
±2
120*
352
438
419
110
92
98
33191
34584
39769
167
162
157
559
676* 663*
83
84
64
± 31
± 39
± 12
±4
± 15
±6
± 3577
± 3185
± 2089
± 10
± 24
±5
± 24
± 21
±9
±6
±1
± 22
321
429* 413*
124
109
106
33731
46622*
30408
178
134*
198
537
620
448
63
58
82*
± 31
± 39
± 25
±6
±3
±6
± 2774
± 4073
± 3590
±6
±4
± 22
± 34
± 66
± 28
±5
±4
±3
249
427* 355*
107
31750
58910*
51289*
211
273*
539*
520
651* 569*
60
63
67
± 10
± 27
± 19
±6
± 3576
± 5335
± 2887
± 13
± 20
± 20
± 16
± 29
±5
±3
±5
148* 172* ±7
±4
± 23
160
305* 303*
183
142
134*
20871
40229*
43391*
181
229*
220*
388
539* 500*
52
71*
64
± 12
± 18
±6
± 28
±5
± 1536
± 5081
± 1779
±3
± 14
±9
± 11
± 30
± 17
±2
±2
±6
228
303* 354*
180
165
152
44062
44047
39521
299
271
229*
555
586
675*
56
63*
56
± 22
±7
± 19
± 3456
± 1359
± 1715
± 10
±4
±7
± 10
± 11
± 17
±2
±2
±3
± 17
±9
± 17 ±7
* P ≤ 0.05 versus control group.
463
464 Because of lack of side effects on the gastrointestinal tract and mucosa damage, AGE seems to be very safe for sows, especially when considering the necessity for 2 months lasting administration. Moreover, considering administration of the investigated substances to pregnant and lactating sows, the proposed approach for the modulation of the development of the gastrointestinal tract of piglets appears to be very safe and convenient. Horie et al. (6), observed that due to the reduction of small intestine damage, AGE diminished weight loss of rats exposed to experimental treatment with antitumour drugs such as methotrexate and 5-fluorouracil. However, morphological parameters of villi were not investigated in these studies. Feldberg et al. (4) and Sivam (11) showed that allicin induced its antimicrobial activity mainly by immediate and total inhibition of RNA synthesis, although DNA and protein synthesis were also partially inhibited, suggesting that RNA was the primary target of allicin action. Lipids contained in bacterial cell membranes facilitate the penetration of allicin and its antibacterial action increases in accordance to higher amount of lipids in the cell membrane. In contrast to Gram-positive Staphylococcus aureus that contains only 2% of cell membrane lipids, antibacterial action of allicin on Escherichia coli, containing 10 fold more lipids in cell membrane, is very high (11). Thus, the possible inhibition of bacterial growth in the intestine of piglets, induced by allicin or its metabolites, may be responsible for improved morphology of villi. In conclusion, increased surface of the small intestine as well as positive effects of allicin and AGE on specific and non-specific defense mechanisms of piglets may be postulated as factors responsible for their improved systemic development (3, 12, 13). Both AGE and allicin may be proposed as good alternatives for antibiotics as feed additives. Regardless of mechanisms of the action of AGE and allicin in relation to these processes, the obtained results of our studies confirmed that improved nutrition at early stages of postnatal development and as the major intrauterine environmental factor, may give advantage not only just after the birth but may have lifelong consequences leading to permanent changes of the structure, physiology, and metabolism of offspring. Considering prenatal life, this phenomenon is termed as “foetal programming” of organism development (15). It can be concluded that maternal administration of the garlic preparations regulates systemic development of piglets. Moreover, AGE and allicin are good candidates for substances affecting foetal programming of systemic development in piglets. However, to support this hypothesis, further investigations in relation to other organs or systems of the organism should be performed.
Acknowledgments: This work was supported by Grant No. PBZ-KBN-093/P06/2003 from the State Committee for Scientific Research (KBN, Poland). References 1. Amagase H., Petesch B.L., Matsuura H., Kasuga S., Itakura Y.: Intake of garlic and its bioactive components. J Nutr 2001, 131, 955S-962S. 2. Ankri S., Mirelman D.: Antimicrobial properties of allicin from garlic. Microbes Infect 1999, 1, 125-129. 3. Dudek K., Tatara M.R., Śliwa E., Siwicki A., Łuszczewska-Sierakowska I., Zipser J., Krupski W., Studziński T.: Effects of perinatal administration of aged garlic extract (AGE) and allicin on non-specific and specific defence mechanisms in sows. Pol J Environ Stud 2005 Suppl II, 14, 69-72. 4. Feldberg R.S., Chang S.C., Kotik A.N., Nadler M., Neuwirth Z., Sundstrom D .C., Thompson N.H.: In vitro mechanism of inhibition of bacterial growth by allicin. Antimicrob Agents Chemother 1988, 32, 1763-1768. 5. Fenwick G.R., Hanley A.B.: The genus Allium. Part 2. Crit Rev Food Sci Nutr 1985, 22, 273-377. 6. Horie T., Awazu S., Itakura Y., Fuwa T.: Alleviation by garlic of antitumor drug-induced damage to the intestine. J Nutr 2001, 131, 1071S-1074S. 7. Hoshino T., Kashimoto N., Kasuga S.: Effects of garlic preparations on the gastrointestinal mucosa. J Nutr 2001, 131, 1109S-1113S. 8. Kaku H., Goldstein I.J., Van Damme E.J.M, Peumans W.: New mannose-specific lectins from garlic (Allium sativum) and ramsons (Allium ursinum) bulbs. Carbohydr Res 1992, 229, 347-353. 9. Matsuura H., Ushiroguchi T., Itakura Y., Hayashi H., Fuwa T.: A furostanol glycoside from garlic bulbs of Allium sativum l. Chem Pharm Bull 1988, 36, 3659-3663. 10. Nagae S., Ushijima M., Hatono S., Imai J., Kasuga S., Matsuura H., Itakura Y., Higashi Y.: Pharmacokinetics of the garlic compound S-allyl cysteine. Planta Med 1994, 60, 214-217. 11. Sivam G.P.: Protection against Helicobacter pylori and other bacterial infections by garlic. J Nutr 2001, 131, 1106S-1108S. 12. Tatara M.R., Śliwa E., Dudek K., Mosiewicz J., Studziński T.: Effect of aged garlic extract (AGE) and allicin administration to sows during pregnancy and lactation on body weight gain and gastrointestinal tract development of piglets. Part I. Bull Vet Inst Pulawy 2005, 49, 349-355. 13. Tatara M.R., Śliwa E., Dudek K., Siwicki A., Kowalik S., Łuszczewska-Sierakowska I., Krupski W., Zipser J., Studziński T.: Influence of perinatal administration of aged garlic extract (AGE) and allicin to sows on some defence mechanisms in their piglets during postnatal life. Pol J Environ Stud Suppl II 2005, 14, 378-381. 14. Weiberg D.S., Manier M.L., Richardson M.D., Haibach F.G.: Identification and quantification of organosulfur compliance markers in garlic extract. J Agric Food Chem 1993, 41, 37-41. 15. Wu G., Bazer F.W., Cudd T.A., Meininger C.J., Spencer T.E.: Maternal nutrition and fetal development. J Nutr 2004, 134, 2169-2172.
